For better diagnosis and management of corneal wound healing problems it is important to understand the basic mechanisms that regulate cellular proliferation and alterations in the phenotypic characteristics of the cells that determine corneal transparency during wound healing. Keratocytes, corneal stromal cells that are quiescent in the normal adult cornea, are activated during wound healing to become proliferative fibroblasts or myofibroblasts with altered phenotypic characteristics. The goal of this project is to elucidate the signaling mechanisms that control and modulate: 1) the cellular proliferation (cell cycle progression) of corneal epithelial cells and activated corneal keratocytes and 2) the phenotypic changes upon the activation of keratocytes. Our previous studies demonstrated that the small GTPase RhoA, through its downstream target ROCK, regulates cell cycle progression in both corneal epithelial cells and activated corneal stromal cells in culture. We have also determined that upon keratocyte activation, the activation of mitogen activated protein kinases (MAPKs), INK and ERK, can lead to downregulation of the expression of several proteins that are critical to the maintenance of corneal transparency. The Specific Aims of this competing renewal proposal test specific hypotheses and predictions about the signaling mechanism in cell cycle regulation in corneal epithelial cells and activated stromal cells. In tissue culture, we will perform functional analyses of the signaling proteins by inhibiting their expression with siRNAs and by modulating their activities with constitutively active or dominant negative recombinant proteins. Using these approaches we will address the following questions: Aim 1) Are mDia, a downstream effector of RhoA, and LIMK, a downstream effector of ROCK, involved in the RhoA/ROCK mediated changes in cell cycle regulatory proteins, cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors (GDIs)?;Aim 2) Does connexin 43, a gap junction protein regulate cell cycle progression?;and Aim 3) Do small GTPases RhoA, Cdc42 and Rac, and the MAPKs, INK and ERK, regulate the expression of keratan sulfate proteoglycans (keratocan and lumican), corneal crystallins (ALDH1 and DTKT) and D3(IV) collagen upon keratocyte activation? This new information will further our knowledge of corneal homeostasis and wound healing mechanisms. It will lead to better diagnoses and treatments of persistent corneal wound healing problems that are related to dysregulated cellular proliferation, and to prevention of nontransparent scar tissue formation following injury, keratoplasty or refractive surgery.